1. Field of the Invention
The invention in the field of peptide chemistry and medicine relates to novel conformationally restricted biologically active peptides and isosteres, methods for producing such peptides based on oxidative or reductive electrochemical coupling reactions, and methods of using such peptides as hormone agonists or antagonists in treating disease.
2. Description of the Background Art
A. Chemical Stabilization of Peptides
New methods for restricting the secondary structure of peptides and proteins are highly desirable for (1) basic structure-function studies, (2) the elucidation of mechanisms, and (3) the rational design of therapeutically useful conformationally-restricted (or "locked") pharmacophores.
These applications are exemplified by an analogue of eel calcitonin, [Asu.sup.1,7 ]-eel calcitonin, in which .alpha.-aminosuberic acid (Asu) replaces the cysteine residues at positions 1 and 7 (Morikawa, T. et al., Experientia 32:1104-1106 (1976)). This analogue had significant biological activity, leading the authors to conclude that the disulfide bond in calcitonin is not essential for biological activity as long as the specific conformation of the peptide is maintained by an intramolecular bridge.
The purely chemical approaches for restricting secondary structure often requires extensive multistep synthetic work (Olson, G. L., J. Am. Chem. Soc. 112:323 (1990)). An alternative approach involves installing covalent bridges in peptides. However, due to the sensitivity of the peptide backbone and side chains, this method necessitates careful protection/deprotection strategies. For example, this problem occurs in the preparation of polymethylene analogues of [Arg.sup.8 ]vasopressin in which .alpha.-aminosuberic acid (Asu) replaces the cysteine residues at positions 1 and 7 and in which the N-terminal amino group is removed (S. Hase et al., Experientia 25:1239-1240 (1969); S. Hase et al., J. Amer. Chem. Soc. 94:3590 (1972)), yielding deamino-dicarba-Arg.sup.8 -vasopressin.
Covalent linkages can, in selected instances, be established using other chemical methods, for example, by lactam formation between carboxylic acid and amine side chains (J. S. Taylor, Acc. Chem. Res. 23:338 (1990)), or by incorporation of pairs of Cys residues which form a disulfide bridge. However, these approaches suffer from disadvantages which the present invention has been designed to overcome in the creation of new cross-linked peptides.
B. Promotion of Peptide Secondary Structures
A number of approaches have attempted to induce .alpha.-helical secondary structures by the introduction of covalent bridges (Taylor, supra). Most of these procedures require an extensive synthetic effort as they involve constructing several intermediates typically, and require orthogonal protection strategies. The potential of intramolecular bridging in peptide design has become evident from recent studies on lactam-bridged amphiphilic structures (Taylor, supra).
For example, after synthesizing a protected peptide linked to an oxime polystyrene-resin through a glutamyl gamma-carboxyl side chain, an internal nucleophilic cleavage step generates the lactam ring and simultaneously frees the protected peptide. The utility of the lactam-bridging approach is exemplified by a 21-residue peptide prepared from three lactam-bridged units in series which exhibits unusually high .alpha.-helicity. This method is, however, cumbersome, and only allows covalent bridging on the hydrophilic side of an amphiphilic structure.
Another method, involving the metallation of an [i,i+4]-bishistidyl or [i,i+4]-histidyl-cysteinyl peptide, raises the .alpha.-helicity of the unmetallated sequence from about 54% to about 90% in 5 mM sodium borate buffer, pH 6.7 at 4.degree. C. (M. R. Ghadiri et al., JACS 112:9633 (1990); S. Marqusee et al., Proc. Natl. Acad. Sci. USA 84:8898 (1987)). The use of toxic or heavy metals (e.g., Cu.sup.+2, Ru.sup.+2, Cd.sup.+2) to induce helicity however makes the method less likely to find application in the design of clinically useful peptides.
C. Electrochemical Methods in Organic Chemistry
Electrochemical methods have a long history in organic chemistry and have recently found preparative application in the field of peptide synthesis (S. Coyle et al., J. Chem. Soc., Chem. Commun. 980 (1976)), largely limited to the installation or cleavage of protecting groups.
Using electrochemical methods, it is possible to convert selectively compounds with functional groups which differ in half-wave potential by approximately 200 mV (M. Baizer, ed., Organic Electrochemistry, 2nd ed., Dekker, New York (1983)). Chemical methods are not readily capable of the precision and extent of variability of redox potential that is essential for this selectivity.
Electrochemical methods generate reactive intermediates in concentration gradients which are highest near the surface of the electrode, whereas solution methods produce low steady-state levels of the intermediate species in the bulk medium (Baizer, supra). The high concentration of reactive intermediate at the electrode results in greater yields of coupling versus reaction with solvent or electrolyte (L. Eberson et al., In: M. Baizer, supra, p. 889; J. H. P. Utley et al., J. Chem. Soc., Perkin Trans. 2:395 (1978)).
One of the classic methods of organic electrochemistry is the Kolbe synthesis (Eberson et al., supra). In this reaction, the anodic oxidation of carboxylic acids produces hydrocarbon coupling products with the loss of carbon dioxide, often with excellent efficiencies. The reaction proceeds under acidic conditions to give free radical intermediates, which react to form either coupling products R--R, EQU 2 RCO.sub.2 H.fwdarw.2 R..fwdarw.R--R
proton transfer, or disproportionation, while under basic conditions the anodic process generates carbenium ion intermediates, which yields nucleophilic addition products (H. -J. Schafer, Top. Cur. Chem., 152:91 (1990)).
By its nature, an electrochemical process involves molecules interacting at an electrode interface at which heterogeneous electron transfer takes place. After becoming loosely associated or perhaps adsorbed on the surface and being exposed to a sufficiently oxidizing or reducing potential, the most reactive functional group present in the molecule will form a reactive intermediate, such as a radical cation, radical anion, or free radical. Proton transfer may then follow, affecting the net charge of the reactive intermediate.
If a second reactive moiety is available at an appropriate distance and located on the same sterically accessible face of the molecule, coupling of the reactive intermediates will lead to bond formation. In the Kolbe method, two carbon free radicals join to form a new macrocyclic ring containing a carbon-carbon single bond. As long as the current density at the electrode is maintained at a sufficiently high level, the probability of forming two reactive intermediates in the same molecule within the lifetime of the first intermediate is reasonably high (Baizer, supra).
D. Electrochemical Reactions of Amino Acids and Peptides
Few precedents exist for the electrochemical transformation of protected amino acids and peptides under typical conditions common to other kinds of molecules (Schafer, supra). Suberic acid derivatives, from which dicarba analogues of cystine-containing peptides have been obtained chemically (for example, oxytocin, calcitonin and somatostatin) have been prepared by Kolbe electrolysis of protected D-Glu and L-Glu (R. F. Nutt et al., J. Org. Chem. 45:3078 (1980)). There, the electrochemical reaction was performed on amino acids, not on a peptide. Electrochemical reactions of amino acids and peptides have been carried out at platinum, lead or glassy carbon electrodes, in solvents routinely used for organic electrochemistry such as methanol, acetonitrile, tetrahydrofuran, and N,N-dimethylformamide (Baizer, supra).
Classical studies by Takayama of the oxidation of simple amino acids in aqueous acid solutions, revealed that anodic electrolysis yields aldehydes resulting from loss of the carboxylic acid and primary e-amino groups. At alkaline pH, oxidation of amino acids yields nitriles as well as aldehydes, depending on the electrode used (Baizer, supra). These side reactions may have prevented the more widespread use of electrochemical reactions in peptide chemistry.
E. Peptide Hormones and Growth Factors
Peptide hormones are central to the regulation of metabolism, differentiation, proliferation, and growth. The relationship between peptide structure and activity remains best understood from studies of synthetic analogues designed to model biologically functional regions of the peptide. Direct structure-function correlation is rare due to difficulty in preparing crystals of intermediate-sized peptides of quality adequate for X-ray study.
There is a long-standing need in the art for a better understanding of how the conformational structure of a peptide modulates its regulatory role, in order to improve the prospect of treating or ameliorating diseases associated with defects or dysregulation of proteins such as growth factors and peptide hormones. A wide range of diseases, including cancer, osteoporosis, diabetes, and other metabolic defects, await the development of rationally designed agonists and antagonists to native polypeptide hormones. As chemical and electrochemical manipulation of peptide structure becomes a more precise science, the likelihood improves for major strides in the prevention and management of a multitude of diseases related to metabolism, cell development and differentiation.
1. Insulin and Insulin-Like Growth Factors
The development and application of insulin for the treatment of diabetes mellitus, the first example of a peptide pharmaceutical, is one of the great medical achievements of the twentieth century. Insulin is a 6 kDa peptide hormone made up of two chains, A and B, linked by a pair of disulfide bonds, and is derived biosynthetically from proinsulin which consists of A and B chains coupled by a third segment, designated C.
Glucose regulation, which is tightly correlated with normal development, is known to be associated also with other insulin-like peptide hormones (P. D. Gluckman, Oxford Rev. Reprod. Biol. 8:1 (1986)). Specific polypeptide hormone growth factors, like insulin, are now known to be critical in development (S. Heyner et al., In: Growth Factors in Mammalian Development, I. Y. Rosenblum et al. (eds), CRC Press, Boca Raton, Fla., 1989, pp. 91-112). The insulin-like growth factors, IGF-I (A. Ullrich et al., EMBO J. 5:2503 (1986)) and IGF-II (E. Rinderknecht et al., FEBS Lett. 89:282 (1978))), have extensive sequence homology with proinsulin. Computer modelling purports a similarity in the tertiary structures of IGFs and insulin (T. L. Blundell et al., Nature 287:781 (1980); T. L. Blundell et al., Feder. Proc. 42:2592 (1983)). X-ray crystal data are not yet available for IGF-I or IGF-II. All of these peptides have overlapping functions with differing activities at each other's receptor.
Insulin has also been found to play a central role in growth regulation as well (D. S. Straus, Endocrinol. Rev. 15:356 (1984)). Receptors which bind insulin and IGFs have been detected in early mammalian embryos. Both deficits and excesses of insulin have been correlated with birth defects (D. E. Hill, In: The Diabetic Pregnancy: A Perinatal Perspective, R. Merkatz et al., (eds) Grune & Stratton, New York, 1979, pp. 155-156). Nanomolar concentrations of insulin and IGF-I induce myoblast differentiation in chick embryos (C. Schmidt et al., FEBS Lett. 116:117 (1983)). Insulin also enhances neuronal proliferation (Garofalo, R. et al., Molec. Cell. Biol. 8:1638 (1988)). IGF-I and insulin are both able to stimulate RNA and protein synthesis (U. Widmer et al., Acta Endocrinol. 108:237 (1985)). Abnormally high levels of insulin or proinsulin have been found to cause abnormal growth, teratogenic effects, and death in chick embryos, possibly by interaction at the IGF receptor (F. DePablo et al., Diabetologia 28:308 (1985)).
2. Melanocyte Stimulating Hormone
Melanocyte stimulating hormone (MSH) is produced in the pituitary, and controls skin melanin dispersion (T. K. Sawyer et al., Am. Zool. 23:529 (1983); E. Schroder et al., In: The Peptides: Synthesis, Occurrence, and Action of Biologically Active Polypeptides, vol. 2, Academic Press, New York, pp. 165ff (1966)). The alpha, beta, and gamma forms of MSH are derived from the precursor proopiomelanocortin, which is also the source of adrenocorticotrophic hormone (ACTH) and the opioid peptide .beta.-endorphin. MSH is thought to play a role in fetal growth and development (Sawyer et al., supra). For example, MSH has been detected in human and other mammalian fetal pituitary tissue (A. J. Kastin et al., Acta Endocrinol. 58:6 (1968)), and postulated to be central to the timing of human birth (R. E. Silman et al., Nature 260:716 (1976)). Fetal MSH is also important for prenatal growth (G. J. Boer et al., Applications of Behavioral Pharmacology in Toxicology, Zbinden et al. (eds), Raven Press, New York, p. 251, 1983). Removal of the fetal rat pituitary prevents the normal growth spurt between days 19 and 21, which can only be restored by exogenous administration of MSH. In addition, treatment with antibodies to MSH stunts the growth during this same interval. The importance of restricted conformation to function in MSH, as has been shown for other cyclic lactam analogues (see below), makes it a good system in which to apply the approaches of the present invention. To date, effective MSH receptor agonists and antagonists have depended on the use of D-amino acids, as in [Nle.sup.4,D-Phe.sup.7 ]-.alpha.-MSH (Schroder et al., supra; W. M. Westler et al., J. Amer. Chem. Soc. 110:6256 (1988)).
3. Cholecystokinins
The C-terminal peptide of cholecystokinin, known as CCK-8 (residues 26-33), is a hormonal regulator of pancreatic secretion and gallbladder contraction, as well as a neuropeptide (Charpentier, B. et al., Proc. Natl. Acad. Sci. USA 85:19681972 (1988)). Cyclization of the CCK-8 analogue, Boc-[2-aminohexanoic acid]CCK-(27-33), has been carried out using a fragment condensation method (Charpentier, B. et al., J. Med. Chem. 30:962-968 (1987)) in conventional chemical peptide synthesis. Two cyclic compounds having an internal amide bond between the side chain amino group of D-Lys-29 and either the .beta.-carboxyl group of a D-Asp-26 residue or the .alpha.-carboxyl group of a D-Glu-26 residue were shown to have high affinity and selectivity for guinea pig brain CCK receptors.